During the last funding cycle, we demonstrated that HD-Ads are promising vectors for liver-directed gene transfer. However, before we push for clinical trials, it is imperative that we generate the most efficient HD-Ads, develop protocols that will allow the use of low-dose HD-Ads to minimize toxicity, and construct regulatable HD-Ads to enhance its utility. To these ends we propose 4 Specific Aims for this Project. In Specific Aim 1 we will produce three different LDL receptor (LDLR) HD-Ads and test them in LDLR-/- mice. Although HD-Ad has a large cloning capacity of up to 37kb, the native human LDLR is still too large to be inserted into these vectors in its entirety. We will produce different LDLR constructs, including a cDNA construct and two chimeric/cDNA constructs, and test their efficacy and toxicity in LDLR-/- mice. In Specific Aim 2, we take this approach a step further and test the most efficacious of these HD-Ad-LDLR constructs in rhesus monkeys with heterozygous LDLR deficiency. We will further test a protocol developed in a Project that first depletes Kupfer cells in these monkeys before HD-Ad-LDLR treatment, an approach that may enahnce the efficacy and reduce the toxicity of HD-Ads. In Specific Aim 3, we will compare the potential lipid lowering and antiatherogenic effect of HD-Ad-LDLR, apoAI or the two vectors together. We will test whether treatment with a dual LDLR+AI transgene vector produces additive or synergistic effects compared with the individual vectors alone; use of a dual transgene vector may reduce potential toxicity by 50% as the two trasgenes will be delivered in a single vector. In Specific Aim 4, we will use HD-Ads that contain a glucose 6 phosphatase (G6Pase) gene to treat mice and dogs that have with GSD-la (G6Pase deficiency). Three different constructs, a cDNA construct, a genomic construct and a regulatable construct, will be compared. The amelioration of the signs and symptoms of GSD-la and the prevention of long-term complications of the disease may require a substantially lower dose of HD-Ads than that needed for the treatment of dyslipidemia. GSD-la may turn out to be an ideal disease target for the application of HD-Ads in the first clinical trials. Information generated from this project together with that from Projects 2-4 will pave the way toward clinical trials using HD-Ads, a highly efficient gene transfer vector for the treatment of genetic disorders.